Marziale Milani

ATPase and ATPsynthetase activity in myosin exposed to low power laser and pulsed electromagnetic fields

Abstract Myosin can interact with actin in the presence of ATP and Ca2+. The resulting association gives rise to an actomyosinic complex which is involved in muscle contraction. Purified myosin contains the enzyme ATPase which catalyses the breakdown of ATP (in ADP plus inorganic phosphate (Pi)). This fundamental enzymatic mechanism is involved in transducing chemical into kinetic energy through the actomyosinic complex. The myosin ATPase activity can be inactivated by chemical (3 M urea, CO2) or physical agents (thermal shocks). Partial recovery of the inhibited activity has been obtained by exposure to low power laser radiation (GaAs and He-Ne) or weak magnetic pulsed fields. The myosin ATPase reverse reaction (ATP synthesis) occurs by incubating myosin ATPase in the presence of ADP (excess). Pi and a system for H+ production (lactate dehydrogenase, lactate, NAD+). Again the enzymatic activity is increased by exposure to low power laser fields or weak magnetic pulsed fields.

Focused ion beam techniques for the analysis of biological samples: A revolution in ultramicroscopy?

Focused Ion Beam (FIB) is a novel technique that allows easy target cell selection, fast operation, high resolution, 3D imaging and sample manipulation during imaging. The FIB technique of microscopy and nanomachining, that is widely spread in semiconductor technology, is reshuffled to open new horizons in the field oflife sciences at cellular and subcellular level. FIB is a source ofions which can be precisely oriented and focused on the sample, supported also by an electron source. The resolution can be as low as I 5nm. Two different operation modes are available: cutting and etching operations of very high precision and at the same time the resulting secondary ions and electrons provide follow up sample imaging. FIB tomography ability has been tested to provide information on cell characterisation, cell division time sequence, view of inner structures and investigation of membrane structural properties. We compare the performances and the advantages of different high-resolution microscopy techniques: Soft X-ray Contact Microscopy (SXCM), Focused Ion Beam (FIB) and Transmission Electron Microscopy (TEM). These have been used to image Saccharoniyces cerevisiae yeast cells, a rather well known sample of great biological interest. TEM is an established technique used as a benchmark for comparison.

Magnetic field effects on human lymphocytes

The results are discussed of a systematic investigation into the electromagnetic field (EMP) exposure consequences on human lymphocytes. These artificial fields have intensities comparable to the Earths magnetic field, and are used for exposures up to 4 days. Different and complementary techniques are used to safely assess the consequences of EMFs on the cells; in particular, morphology, metabolism, and population dynamics are investigated. The recourse to ultramicroscopy, pressure monitoring in sealed bottles, atomic mass spectroscopy, and cytofluorimetry techniques give good insight into the EMF-induced changes. A statistically significant deviation of irradiated samples with respect to control samples is reported. A critical analysis and a survey of similar experiments reported in the literature led us to examine the experimental setup with attention to the geometry of the irradiation system. Yeast cells were used as a model system to statistically test the different steps in the overall procedure, thanks to information gathered during a radiobiology experiment performed at the Rutherford Appleton Laboratory. Finally, the role of different magnetic field detectors in the reproducibility of the experiments is carefully discussed.

Quantum mechanics: a breakthrough into biological system dynamics

Abstract Superconductive and Josephson junction behaviour may be present in living cells. Positive experimental evidence is discussed. It is shown that the “Josephson junction paradigm” makes the connection possible between many different experimentally observed properties of living matter, allowing a further step towards a unitary framework for both physical and biological systems. Electromagnetic interaction plays a fundamental role. Superconductivity in biological systems can be demonstrated on theoretical grounds starting from a quantum microscopic approach to their dynamics. Josephson-like behaviour can be expected (and is observed) in single cells at cytokinesis or for two nearby cells. Electromagnetic interaction between cells is a well-endowed candidate for driving intercellular communication and for organizing the transport of material through gap junctions established between adjacent cells.

Focused ion beams and life science applications: cell tomography and biomachining at ultrahigh resolution

A new technique of Focused Ion Beam (FIB) microscopy- nanomachining is proposed for life sciences. Its performances are compared with those of currently available ultramicroscopy apparatuses. Ultra-high resolution tridimensional tomography can be performed on whole cells without preparation. This can be achieved by sequentially etching layers of material and subsequently viewing the result of the operation under a different perspective. Very fast imaging times (minutes) allow quasi real time microscopy. The complementary technique of nano-biology can be performed on the same apparatus. The use of the ion beam allows to imaging both the surface and the inner part of the sample along any desired plane that can be chosen while the observation is on.

Lasing properties and nonlinearities of dyes under high power pumping

Nitrogen lasers have been used for many years to make dye solutions lase. A nitrogen laser, which transverse electrical discharge in gas at atmospheric pressure has been built in our laboratory. It has been characterized and applied to pump different dyes: Rhodamine 6G, Coumarin 440, DOTCI, and pyranine in simple on axis geometric configuration. It has been shown that pyranine can lase in the absence of any optical external mirror cavity, this happens at very low threshold, and in different solvents. Dyes under consideration can be grouped into two major classes according to their lasing behavior independently on their concentration in the solvent: Rhodamine 6G and DOTCI can lase both axially or transversally and Coumarin 440 and pyranine can lase only axially. Other intriguing features have been observed that span from simultaneous multiple beam generation, to super fluorescence and to distribute axial pumping of dye solutions. A preliminary basis for understanding and controlling such processes is the spatial energy distribution and the energy density of the beam.

X-ray irradiation of yeast cells

Saccharomyces Cerevisiae yeast cells were irradiated using the soft X-ray laser-plasma source at Rutherford Laboratory. The aim was to produce a selective damage of enzyme metabolic activity at the wall and membrane level (responsible for fermentation) without interfering with respiration (taking place in mitochondria) and with nuclear and DNA activity. The source was calibrated by PIN diodes and X-ray spectrometers. Teflon stripes were chosen as targets for the UV laser, emitting X-rays at about 0.9 keV, characterized by a very large decay exponent in biological matter. X-ray doses to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. After irradiation, the selective damage to metabolic activity at the membrane level was measured by monitoring CO2 production with pressure silicon detectors. Preliminary results gave evidence of pressure reduction for irradiated samples and non-linear response to doses. Also metabolic oscillations were evidenced in cell suspensions and it was shown that X-ray irradiation changed the oscillation frequency.

Biosystem response to soft-X-rays irradiation: non-monotonic effects in the relevant biological parameters of yeast cells

SummarySoft-X-rays irradiation of yeast cells allows selective interference with different cellular structures. The monitoring of different physical parameters leads to substantial variations in the response to X-rays showing that monotonicity should not be taken for granted.

Feedback effects in optical communication systems: characteristic curve for single-mode InGaAsP lasers

An experimental analysis of InGaAsP injection lasers shows an unexpected decrease of the differential quantum efficiency as a function of injected current when optical power is fed back into the active cavity of a diode inserted into a long transmission line. To investigate the response of laser diodes to optical feedback, we base our analysis on a microscopic model, resulting in a set of coupled equations that include the microscopic parameters that characterize the material and the device. This description takes into account the nonlinear dependence of the interband carrier lifetime on the level of optical feedback. Good agreement between the analytical description and experimental data is obtained for threshold current and differential quantum efficiency as functions of the feedback ratio.

An ensemble of new techniques to study soft-X-ray-induced variations in cellular metabolism

An ensemble of new techniques has been developed to study cell metabolism. These include: CO2 production monitoring, cell irradiation with soft X rays produced with a laser-plasma source, and study of oscillations in cell metabolic activity via spectral analysis of experimental records. Soft X-rays at about 0.9 keV, with a very low penetration in biological material, were chosen to produce damages at the metabolic level, without great interference with DNA activity. The use of a laser-plasma source allowed a fast deposition of high doses. Monitoring of CO2 production allowed us to measure cell metabolic response immediately after irradiation in a continuous and non invasive way. Also a simple model was developed to calculate X-ray doses delivered to the different cell compartments following a Lambert-Bouguet–Beer law. Results obtained on Saccharomyces cerevisiae yeast cells in experiments performed at Rutherford Appleton Laboratory are presented.